Rates of spontaneous cleavage of glucose, fructose, sucrose, and trehalose in water, and the catalytic proficiencies of invertase and trehalas

Article abstract of DOI:10.1021/ja802206s

The half-lives for spontaneous hydrolysis of trehalose and sucrose at 25 °C are 6.6 × 10⁶ years and 440 years. The half-lives for decomposition of the hydrolysis products glucose and fructose are 96 years and 70 days, respectively. Whereas sucrose and trehalose differ by a factor of 15000 in their rates of uncatalyzed hydrolysis, the reactions catalyzed by invertase (EC 3.2.1.26) and trehalase (EC 3.2.1.28) proceed at similar rates. Accordingly, the attainments of invertase as a catalyst are modest, but the rate enhancement and catalytic proficiency produced by trehalase approach the high levels achieved by polysaccharide hydrolases. Copyright

Full text of DOI:10.1021/ja802206s

Published on Web 05/28/2008
Rates of Spontaneous Cleavage of Glucose, Fructose, Sucrose, and
Trehalose in Water, and the Catalytic Proficiencies of Invertase and Trehalas
Richard Wolfenden* and Yang Yuan
Department of Biochemistry and Biophysics, UniVersity of North Carolina, Chapel Hill, North Carolina 27599
Received March 25, 2008; E-mail: water@med.unc.edu
Most of the early experimental work on enzyme kinetics was
based on the properties of yeast invertase (ꢀ-fructo-furanosidase,
1
EC 3.2.1.26), whose robust activity (together with the availability
of a continuous polarimetric assay) enabled Michaelis and Menten
to establish the existence of a quantitative relationship between the
2
rate of an enzyme reaction and the concentration of its substrate.
To appreciate the proficiency of an enzyme as a catalyst, it is also
desirable to have information about the relative rates of the catalyzed
3
and uncatalyzed reactions. Surprisingly, the literature discloses no
report of the rate of spontaneous hydrolysis of sucrose (although
4
there have been many studies of the acid-catalyzed reaction). That
information would be of special interest in view of the remarkable
7
5
resistance to hydrolysis (t1/2 ∼10 y), of the 1-4 “head-to-tail”
glycosidic linkages that join the common glucose polymers cel-
lulose, chitin, amylose, and glycogen. Thus, polysaccharide hy-
drolases (e.g., ꢀ-amylase) generate the largest rate enhancements
that are known to be produced by hydrolytic enzymes that operate
6
without the assistance of metals or other cofactors. Here, we
Figure 1. Arrhenius plots for hydrolysis of sucrose and trehalose (0.05
M) in 0.1 M potassium phosphate buffer, pH 8.1.
describe the uncatalyzed hydrolysis of sucrose and trehalose, in
which the constituent monosaccharides are symmetrically joined,
by a “head-to-head” glycosidic linkage between their carbonyl
groups (Chart 1).
Chart 1. Nonreducing Disaccharides
Samples of sucrose or trehalose (0.05 M) were incubated in
potassium phosphate and carbonate buffers (0.1 M) in Teflon-lined
acid digestion bombs (Parr Instrument Co. #276AC) at elevated
temperatures in Barnstead-Thermolyne Corp. #47900 ovens, moni-
toring the temperature of the sample with an ASTM mercury
thermometer. In experiments conducted at pH values below 9,
samples were sealed under argon in quartz tubes (3 mm ID, 4 mm
OD). At timed intervals, samples were cooled and diluted 50-fold
2
with D O to which pyrazine had been added as an internal
integration standard. Proton NMR spectra were obtained using a
Varian 500 MHz NMR spectrometer equipped with a cold probe.
The decomposition of sucrose, trehalose, and glucose was monitored
by following the disappearance of the anomeric proton resonances
at ∼4.2 ppm, while the decomposition of fructose was followed
Table 1. Rate Constants and Activation Parameters for the
Uncatalyzed Decomposition of Mono- And Disaccharides
q
q
q
-
1
)
∆G 25C
∆H
(kcal/mol) (kcal/mol) (kcal/mol)
T∆S 25C
k25C (s
7
-11
by monitoring the disappearance of a multiplet at 3.5 ppm. In
sucrose
trehalose
R-methylglucopyranoside
anhydrocellobiitol
glucose
fructose
5 × 10
3.3 × 10
2 × 10
31.4
37.1
37.4
37.9
30.5
26.8
27.3
32.0
30.3
35.0
28.0
15.9
-4.1
-5.1
-7.1
-2.5
-2.5
-10.9
-
15
potassium phosphate (0.1 M, pH 8.1) or potassium carbonate buffer
a
-15
-15
-10
-7
(
0.1 M, pH 10.3), the decomposition of each of these molecules
b
1 × 10
followed simple first order kinetics to at least 95% completion under
all conditions examined.
2.3 × 10
1.1 × 10
For sucrose, trehalose, glucose, and fructose, the rates of
decomposition observed at various temperatures yielded linear
Arrhenius plots (Figure 1) with a least-squares regression coefficient
a
b
Reference 5. Reference 15.
(0.1 M, pH 8.1) are shown in Table 1. As in the case of
2
5
of r g 0.97 and n g 7. But decomposition of the reducing
1-methylglycosides, acid catalysis was observed at low pH, but
disaccharides lactose, cellobiose, and maltose yielded Arrhenius
plots that were concave upward, consistent with the availability of
alternate routes for decomposition, including aldol cleavage and
oxidation (Supporting Information).
Rate constants and activation parameters observed for the
hydrolysis of trehalose and sucrose, in potassium phosphate buffer
rates of hydrolysis did not vary significantly in the pH range
between 7 and 12. Rates of hydrolysis of sucrose and trehalose did
not change with changing ionic strength in the range from 0.2 to 2
M KCl, and did not vary significantly with phosphate buffer
concentrations varying between 0.065 and 1.2 M. The extrapolated
rate constant for the uncatalyzed hydrolysis of trehalose at 25 °C
7
548 9 J. AM. CHEM. SOC. 2008, 130, 7548–7549
10.1021/ja802206s CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Kinetic Parameters for Invertase and Trehalase, pH 8.1
invertase
a
trehalase
(honeybee)
ꢀ-amylase
c
(sweet potato)
b
(
yeast)
kcat (s-
1
)
1.0 × 10
2.5 × 10
4.2 × 10
5 × 10
4
2.6 × 10
3
1.4 × 10
3
-
5
2
-4
6
-5
Km (M)
6.6 × 10
3.9 × 10
3.3 × 10
1.2 × 10
7 × 10
kcat/Km (s M-1
-
1
)
2 × 10
22
7
-
1
-11
-15
21
-15
knon (s
(
)
1.9 × 10
-
1
15
kcat/Km)/knon (M
)
8 × 10
10
a
b
c
Reference 16. Reference 17. Reference 5.
6
corresponds to a half-life of 6.6 × 10 years, not very different
from the rate of hydrolysis reported earlier for R-methylglucopy-
5
ranoside (Table 1). But the hydrolysis of sucrose was found to
proceed much more rapidly, with t1/2 ) 440 years at 25 °C (Table
1
).
Figure 2. Rate constants for uncatalyzed biological reactions in water (for
Decomposition of the product monosaccharides (fructose and
references, see ref 6).
glucose) was found to occur much more rapidly than disaccharide
hydrolysis, accounting for their failure to appear in more than
fleeting amounts during disaccharide hydrolysis. Experiments
conducted on glucose (0.05 M) under an argon atmosphere over
the temperature range between 70 and 130 °C showed that glucose
1
4
diester monoanions appear to be more difficult than glycoside
cleavage (Figure 2). Phosphate ester hydrolyses are catalyzed by
enzymes containing metal ions that are capable, by themselves, of
acting as strong catalysts. But glycoside hydrolases act as purely
protein catalysts, and these enzymes appear to be matched only by
OMP decarboxylase in their ability to catalyze very difficult
‡
‡
-1
decomposes with ∆H ) 28.0 kcal/mol and ∆S ) -0.4 cal deg
-1
mol . The extrapolated rate constant for glucose decomposition
-
10 -1
in neutral solution at 25 °C was 2.3 × 10
s
(t1/2 ) 96 years),
3
reactions without the assistance of metals or other cofactors.
with a corresponding t1/2 of 70 days for fructose (Supporting
Information).
In summary, the intrinsic stabilities of sucrose, trehalose, glucose,
and fructose in water have been measured for the first time. Because
sucrose is so much more readily hydrolyzed than trehalose, the
attainments of invertase as a catalyst are relatively modest. But
the rate enhancement and catalytic proficiency produced by trehalase
approach the very high levels achieved by the polysaccharide
hydrolases.
Trehalose cleavage occurs at the C-1 atom of one of its
symmetrically situated glucopyranosyl moieties, at a rate that is
the same, within a factor of 2, as the rate of cleavage of
R-methylglucopyranoside (Table 1). Thus, the effect of the leaving
group (methoxide vs glucopyranoside) is slight. The 15000-fold
more rapid hydrolysis of sucrose suggests that sucrose cleavage
occurs at another site, presumably at the fructofuranosyl moiety.
The acid-catalyzed hydrolysis of ꢀ-methylfructopyranoside, which
Acknowledgment. This work was supported by NIH Grant No.
GM-18325.
8
involves cleavage of the fructosyl-oxygen bond is known to
4
proceed ∼10 -fold more rapidly than that of R-methylglucopyra-
Supporting Information Available: Arrhenius plot of rate constants
for decomposition of fructose and glucose; rate constants observed for
the decomposition of lactose, celloboise, and maltose. This material is
available free of charge via the Internet at http://pubs.acs.org.
9
noside. These differences in reactivity are probably related to the
greater stability of a tertiary oxocarbenium ion generated at C-2 of
fructose than that of the secondary ion generated at C-1 of glucose;
and their similar magnitudes suggest that carbocationic character
may have developed to a similar extent in the transition states for
the spontaneous and the acid-catalyzed reactions.
Whereas there is a 15000-fold difference between sucrose and
trehalose in their rates of uncatalyzed hydrolysis, the hydrolytic
reactions catalyzed by invertase (EC 3.2.1.26) and trehalase (EC
References
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(
(
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(
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3
.2.1.28) exhibit similar values of kcat and K
m
(Table 2). Thus, the
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(
7) Blanchard, J. S.; Brewer, C. F.; Englard, S.; Avigad, G. Biochemistry 1982,
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2
1, 75–81.
(
(
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1
0
of attack. Accordingly the trehalase-catalyzed reaction probably
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(
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(
(
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of trehalase can then be estimated in the usual manner (knon/(kcat
/
-21
-1
-22
K
m
)), as 8 × 10
M , not very different from the value (10
5
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M) for ꢀ-amylase, another “inverting” enzyme. But in the case of
invertase, the catalyzed and uncatalyzed reactions proceed by
different mechanisms, rendering estimation of transition state
affinity in the usual sense problematic.
Of the unusually slow reactions that are catalyzed by enzymes,
(
(
1
1,12
6
5, 2657–2665.
1
3
only the hydrolyses of phosphoric acid monoester dianions and
JA802206S
J. AM. CHEM. SOC. 9 VOL. 130, NO. 24, 2008 7549